This invention claims priority of a German filed patent application DE 199 49 272.7
The present invention concerns a confocal laser scanning microscope having at least one laser light source for illuminating a specimen and at least one detector for detecting the detected light coming from the specimen.
Laser scanning microscopes of the generic type have been known for some time, and are used, inter alia, in the semiconductor industry for wafer inspection and in biomedical basic research. Single-mode lasers that are capable of generating a diffraction-limited intensity distribution in the focal plane of a microscope objective are used as the light source for confocal laser scanning microscopy (CLSM). For this purpose, the laser light of a single-mode laser is usually focused onto a small illumination aperture having a diameter of approx. 100-300 xcexcm, so that this illumination aperture constitutes the single-point light source of the CLSM.
Light sources that are not single-mode lasers cannot create confocal illumination that has sufficient illumination density in the form of a diffraction-limited intensity distribution at the microscope objective focus. The emitted light intensity of a spatially extended non-single-mode laser light source cannot be focused well enough onto such a small illumination aperture, so that the resulting illumination density at the microscope objective focus is too low for most CLSM applications. Merely by way of example, reference is made to U.S. Pat. No. 5,578,818, in which an LED (light-emitting diode) is used as the light source for a CLSM and in which these problems occur.
Depending on the CLSM application, lasers whose wavelength regions extend from the UV to the IR region are used. In particular, lasers that are suitable for CLSM microscopy and that emit light in the UV region are generally of considerable overall size and require a very particular laboratory structure, for example for a complex water-cooled circulation system. The high acquisition and operating costs of such a UV laser system are a particular barrier to extensive use.
It is therefore the object of the present invention to configure and develop a confocal laser scanning microscope of the generic type in such a way that light sources with low acquisition and operating costs can also be used for CLSM applications.
The aforesaid object is achieved by the features of claim 1. According to this, a confocal laser scanning microscope is characterized in that a light source is provided that is not a single-mode (TEM00) laser light source; and that in order to attain a sufficient signal-to-noise ratio in the image data of the specimen, the system parameters of the laser scanning microscope are adjustable.
What has been recognized according to the present invention is firstly that by coupling alternative light sources into a CLSM, the signal-to-noise ratio of the measured image data does not allow data analysis in the usual sense because of the low illumination density of the intensity distribution in the focal plane of the microscope objective. The system parameters of a CLSM can, however, be adjusted in such a way that the use of an additional light source is possible, with a sufficient signal-to-noise ratio in the image data. Suitable system parameters might be, for example, the diameter of the illumination or detection pinhole, the recording duration, or the gain of the detection signal.
The additional light source can be operated simultaneously with the laser light source of the CLSM for data recording. As a result, the specimen properties in terms of the light from the laser light source and the additional light source can be detected simultaneously, if suitable filters or beam splitters are present for the purpose in the beam path of the CLSM. If no corresponding filters/beam splitters are used, then the additional light source can be operated as an alternative to the laser light source. In this context, it might be possible first to perform a data recording that detects the specimen properties with the laser light source, and then to perform a data recording using the additional light source.
In a concrete embodiment, the detected light is detectable in accordance with the confocal principle. The detected light of the specimen is thus detected confocally when the specimen is illuminated both with the laser light source and with the additional light source. Since the detected light of the specimen passes along the same optical beam path when illuminated both with a laser light source and with an additional light source, this ensures that the image data recorded in this fashion are coincident in terms of their spatial coordinates (co-localization).
In very general terms, it is conceivable for the additional light source to be configured as a metal halide vapor lamp, as a discharge lamp, or as an arc lamp. With regard to a specific CLSM application, the additional light source used in each case is to be selected and coupled into the CLSM as a function of the particular properties of the light required for it.
An HBO or XBO lamp could be used as the additional light source.
Advantageously, an HBO lamp is usually already present in confocal fluorescence laser scanning microscopes, since these are often also operated as conventional fluorescence microscopes, so that a corresponding utilization thereof for confocal detection is associated with little additional design outlay and thus with almost no additional cost. In particular, the high light emission in the UV and visible regions makes possible a number of CLSM applications.
A halogen lamp could furthermore be used as the additional light source. On the one hand this could be a halogen lamp that is matched to a specific application; on the other hand the lamp of the conventional microscopexe2x80x94which, like the HBO lamp, is generally present in a CLSMxe2x80x94could be used.
A multi-mode laser could also serve as the additional light source. The use of an LED or an electron beam collision light source is also conceivable.
Light of the additional light source is coupled into the beam path of the CLSM by way of mirrors, lenses, and filters. In this context, lenses can be used to influence the beam properties of the light of the additional light source, and the beam direction of the light of the additional light source can be modified with one or more mirrors and lastly can be coupled with a filter or beam combiner into the beam path of the CLSM. This filter/beam combiner could, for example, be embodied as a dichroic beam splitter.
Advantageously, the coupling of light of the additional light source into the beam path of the CLSM is accomplished by way of an optical fiber. The use of an optical fiber yields the known advantages of an optical element of this kind, namely vibrational decoupling between the CLSM and additional light source, and flexible light transport with no reservations in terms of safety precautions.
Advantageously, light of the additional light source is coupled into the optical fiber without coupling-in optics. Thus there is no focusing optical system, for example in the form of a lens, between the lamp body and the optical fiber end; instead the optical fiber end is positioned directly on the lamp body. All that is necessary for this purpose is to make available a suitable device that establishes and immobilizes the position of the optical fiber end relative to the lamp.
The optical fiber is configured, in terms of the wavelength region of the coupled-in light, as either a single-mode or multi-mode optical fiber. With regard to the total intensity of the light of the additional light source to be coupled in, and the intensity distribution over the cross section of the light beam emerging from the optical fiber, a multi-mode optical fiber may be preferentially used. The latter allows more light to be transported if the beam profile of the light emerging from the optical fiber plays a subordinate role.
In a concrete embodiment, illumination with the additional light source takes place in such a way that a largely point-like intensity distribution exists in the specimen plane of the microscope objective. This kind of illumination could be achieved with a suitable arrangement of mirrors, lenses, illumination apertures, and filters. Ultimately the beam guidance of the light of the additional light source is comparable to that of the laser light source of the CLSM. In particularly advantageous fashion, the point-like illumination is diffraction-limited. This would then result in confocal illumination by way of the additional light source.
In a further embodiment, illumination with the additional light source is implemented as conventional bright-field microscope illumination. The optical system that couples the additional light source into the beam path of the CLSM is configured in such a way that the entire specimen field of the microscope objective can be illuminated with light of the additional light source. In an advantageous embodiment of the invention, the detected light that returns from the specimen in the context of this type of illumination is detected in accordance with the confocal principle using the detector of the CLSM. In comparable fashion, illumination with the additional light source as conventional dark-field microscope illumination is conceivable.
Illumination by the additional light source in the specimen plane could also be accomplished in the form of an illumination pattern. Here again, the size and shape of the illumination pattern of the coupling-in optical system can be shaped by the additional light source. It is thus possible in advantageous fashion, for example by introducing replaceable projection patterns, to vary the illumination pattern in the specimen plane. In a concrete application, the illumination pattern in the specimen plane is circular and has a diameter of 10 xcexcm.
The CLSM equipped with an additional light source can be used, in particular, for fluorescence microscopy. For example, a CLSM having an HBO lamp as the additional light source could be used to excite, with the UV portion of the emission spectrum of the HBO lamp, fluorescent dyes whose absorption spectrum lies in the UV region. As a result, it is unnecessary to acquire an expensive UV laser that occupies a great deal of space and is cumbersome to operate. It is also conceivable to use a CLSM that has a corresponding additional light source to excite fluorescent dyes whose absorption spectrum lies in the visible or IR region.
For basic medical research in particular, FRET microscopy could be performed with a microscope according to the present invention. With this technique, dyes are excited by illumination and emit the excitation energy, or portions thereof, in nonradiative fashion to adjacent dyes, which are thereby in turn excited and emit characteristic electromagnetic radiation. For example, CFP (cyan fluorescent protein) as donor and EGFP (enhanced green fluorescent protein) as acceptor would be suitable. In addition, it would be possible with a microscope according to the present invention to excite fluorescent dyes that are suitable as caged compounds. This application requires excitation light in the UV region, which can be achieved, for example, with an HBO lamp as the additional light source.
In particularly advantageous fashion, in order to attain a usable signal-to-noise ratio in the image data, the specimen is scanned sufficiently often that the image data thereby recorded can be averaged. Since the coupling in of an additional light source generally results in a lower light intensity in the specimen region compared to the laser light source of a CLSM, the signal of the detected light produced by the additional light source is weaker than the detected light produced by the laser light source of the CLSM. Repeated recording of data from the same specimen region allows statistical averaging, which takes into account the smaller detected signal. In the same fashion, the integration time per pixel during scanning of the specimen can be selected accordingly. For example, the integration time per pixel for the detected light produced by the additional light source could be five times as long as for the detected light produced by the laser light source of the CLSM. This would consequently be associated with a fivefold increase in the data recording time.
A usable signal-to-noise ratio could also be attained by selecting the diameter of the detection pinhole. A greater diameter for the detection pinhole results in an elevated signal-to-noise ratio in the detected image data; in this case the spatial resolution of the corresponding data recording is then reduced in accordance with the underlying optical correlations.